This invention was supported in part by funds from the U.S. government (NIH Grant No. HD07796-27) and the U.S. government may therefore have certain rights in the invention.
Glucosamine-6-sulfatase (G6S) is a lysosomal enzyme found in all cells. This exo-hydrolase is involved in the catabolism of heparin, heparin sulphate and keratin sulphate. Deficiencies in G6S result in the accumulation of undegraded substrate and the lysosomal storage disorder mucopolysaccharidosis type IIID.
Regional mapping by in situ hybridization of a 3H-labeled human G6S cDNA probe to human metaphase chromosomes indicated that the G6S gene is localized to chromosome 12 at q14. Localization to the G6S gene to chromosome 12 was confirmed via Southern blot hybridization analysis of DNA from human x mouse hybrid cell lines (Robertson et al. Hum. Genet. 1988 79(2):175-8).
Human liver contains two major active forms of glucosamine-6-sulfatase, form A which has a single 78 kDa polypeptide and form B which has two polypeptides of 48 kDa and 32 kDa. A 1761 base pair cDNA clone encoding the complete 48 kDa polypeptide of form B has been isolated (Robertson et al. Biochem. Biophys. Res. Commun. 1988 157(1):218-24). This sequence reveals homology with the microsomal enzyme steroid sulfatase. The amino acid sequence was also deduced from this human G6S clone (Robertson et al. Biochem. J. 1992 288(2):539-44). The predicted sequence has 552 amino acids with a leader peptide of 36 amino acids and contains 13 potential N-glycosylation sites, 10 of which are believed to be used. The derived amino acid sequence shows strong sequence similarity to other sulfatases such as the family of arylsulfatases.
The present invention relates to the identification and/or cloning of new, evolutionarily conserved members of a subfamily of sulfatases, referred to herein as Sulf-1 and Sulf-2, from quail embryos (QSulf-1), C. elegans (CeSulf-1), Drosophila melanogaster (DmSulf), mice (MSulf-1 and MSulf-2) and humans (HSulf-1 and HSulf-2).
The present invention also relates to Functional Embryonic Technologies (FETs) which serve as convenient and efficient embryo assays for the investigation and determination of the developmental functions of regulatory genes. Using FETs, members of this new family of sulfatases are demonstrated herein to be essential components of Sonic hedgehog (Shh) inductive signaling which is critical for the specification of neural and mesodermal lineages, as well as other lineages in the vertebrate embryo.
Thus, the present invention also relates to compositions and methods of using these compositions to modulate the expression and/or activity of proteins which are members of this subfamily of sulfatases to modify growth and differentiation of cells, as well as viral infection and inflammation. These methods are believed to be useful in the treatment of cancer, including metastases; in inducing differentiation of cells into myoblasts, neural cells and renal cells for use in the treatment of skeletomuscular degenerative diseases, neurodegenerative diseases and renal degenerative diseases; in inhibiting infection via viruses which utilize sulfated heparin proteoglycans for entry into cells; and in controlling the recruitment of lymphocytes by cells to a site of inflammation.